Although in genetic engineering numerous polypeptide expression systems for prokaryotic and eukaryotic hosts are already known, there is a continuing demand for novel systems which have advantages over the known systems.
Very widely used as hosts for the production of polypeptides are yeasts, e.g. Saccharomyces cerevisiae, for which different types of vectors exist.
Integrating vectors which do not contain an autonomously replicating sequence (ARS). These vectors have usually low transformation rates and usually lead to single copy integration into the yeast genome. PA1 Extrachromosomally replicating vectors which can be subdivided in: PA1 providing a yeast expression plasmid comprising a functional CUP1 gene and a polypeptide expression cassette, PA1 transforming a yeast strain that contains not more than one functional CUP1 gene in the genome with said yeast expression plasmid, and selecting transformed yeast cells from untransformed yeast cells.
Vectors containing autonomously replicating sequences (ars vectors). These vectors are usually present in high copy numbers in the cell, however, they are frequently lost during cell division. PA2 Vectors containing a DNA sequence acting as a centromer during cell division (cen vectors). These vectors, though very stable, are present only in a few copies in the cell. PA2 Vectors derived from naturally occurring yeast plasmids, e.g. vectors derived from the two micron-like plasmid (two-micron vectors). Such two micron-derived plasmids occur in high copy number, their stability however is impaired when heterologous DNA is inserted.
There are several possibilities for improving the stability and for regulating the copy number of a vector. In order to achieve this goal it has been suggested to insert the CUP1 gene, coding for a metallothionein, into the vector (U.S. Pat. No. 4,935,350; R. C. A. Henderson et al., Current Genetics 9 (1985), 133-138).
Metallothioneins (MTs) are small, cysteine-rich metal binding polypeptides widely distributed among eukaryotes. S. cerevisiae contains normally a single MT protein that is encoded by the CUP1 gene. The CUP1 locus has been shown to confer copper resistance to yeast cells and consists of e.g. 10 or more tandemly repeated copies of the CUP1 gene. Copper resistance relies on a combination of CUP1 amplification and CUP1 transcriptional induction following the addition of exogenous copper. A cis acting upstream activation site (UAS.sub.c) required for promotion of copper-inducible transcription of the CUP1 gene has been identified as well as the binding of a cellular factor to UAS.sub.c. The binding factor is the product of ACE1 (=CUP2) gene which is essential for copper-induced transcription of CUP1 gene. The ACE1 protein is a transcriptional activator that binds copper ions thereby altering its conformation and activating its DNA-binding domain. The conformational change of the ACE1 protein and its binding to the UAS.sub.c eventually allows the CUP1 gene to be transcribed. An important feature of the CUP1 system is its autoregulation. This depends on the ability of the CUP1 protein (metallothionein) to bind copper ions itself. Thus, the CUP1 protein appears to repress its own synthesis by complexing free copper ions in the cells, which, in turn, interferes with ACE1 activation.
An insertion of a functional CUP1 gene and a polypeptide expression cassette into a two micron-derived plasmid surprisingly leads to enhanced stability of the two micron-derived plasmid and to amplification via an increased plasmid copy number in the presence of copper ions. This is surprising because the two micron-derived plasmid itself contains already all functions for high copy number extrachromosomal existence. The increased plasmid copy number frequently causes an improved expression of heterologous genes also present on the plasmid. It was surprisingly found, that yield in heterologous gene expression can be further increased significantly if the genuine chromosomal CUP1 genes are disrupted, or if only a single copy of the CUP1 gene, that can not be amplified, is present in the genome.